PROJECT SUMMARY/ABSTRACT Hematopoietic stem cell (HSC) transplantation is a curative treatment for many blood diseases and cancers. However, these procedures still need to be optimized to improve patient outcomes and survival. In part, we have been limited by our poor understanding of how donor HSCs interact with the recipient bone marrow after transplantation. This research proposal seeks to address fundamental questions regarding the cellular interactions between HSCs and niche cells. Based on our previous data, we have shown that the niche not only instructs HSC, but that HSC also transmit instructive cues to the niche. These include triggered remodeling of surrounding endothelial cells into a supportive pocket that ?cuddles? the HSC. To better describe this novel cellular structure, we combined multiple imaging modalities to produce the first correlative light and electron microscopy data of an endogenous stem cell in its niche. We have since performed functional genetic studies that provided mechanistic insight into the formation of this structure. These preliminary results have led us to pursue the following Specific Aims: 1) Identify the HSC-derived paracrine signals that mediate EC remodeling to form a niche for HSCs; 2) Identify mechanisms of adhesive HSC-EC niche interactions that promote signaling and stable expansion of the HSC pool. We will perform in vivo genetic knockdown and small molecule treatments, together with live imaging, to understand the dynamic interactions between HSC and endothelial cells. We will then translate these results to an in vitro vascular niche with the potential to expand HSCs prior to transplantation. Ultimately, we hope our studies will lead to improved clinical HSC transplantation protocols.